David Langlais

Interferon Regulatory Factor 8 Regulates Pathways for Antigen Presentation in Myeloid Cells and during Tuberculosis

IRF8 (Interferon Regulatory Factor 8) plays an important role in defenses against intracellular pathogens, including several aspects of myeloid cells function. It is required for ontogeny and maturation of macrophages and dendritic cells, for activation of anti-microbial defenses, and for production of the Th1-polarizing cytokine interleukin-12 (IL-12) in response to interferon gamma (IFNγ) and protection against infection with Mycobacterium tuberculosis. The transcriptional programs and cellular pathways that are regulated by IRF8 in response to IFNγ and that are important for defenses against M. tuberculosis are poorly understood. These were investigated by transcript profiling and chromatin immunoprecipitation on microarrays (ChIP-chip). Studies in primary macrophages identified 368 genes that are regulated by IRF8 in response to IFNγ/CpG and that behave as stably segregating expression signatures (eQTLs) in F2 mice fixed for a wild-type or mutant allele at IRF8. A total of 319 IRF8 binding sites were identified on promoters genome-wide (ChIP-chip) in macrophages treated with IFNγ/CpG, defining a functional G/AGAAnTGAAA motif. An analysis of the genes bearing a functional IRF8 binding site, and showing regulation by IFNγ/CpG in macrophages and/or in M. tuberculosis-infected lungs, revealed a striking enrichment for the pathways of antigen processing and presentation, including multiple structural and enzymatic components of the Class I and Class II MHC (major histocompatibility complex) antigen presentation machinery. Also significantly enriched as IRF8 targets are the group of endomembrane- and phagosome-associated small GTPases of the IRG (immunity-related GTPases) and GBP (guanylate binding proteins) families. These results identify IRF8 as a key regulator of early response pathways in myeloid cells, including phagosome maturation, antigen processing, and antigen presentation by myeloid cells.

Irf8-regulated genomic responses drive pathological inflammation during cerebral malaria.

Interferon Regulatory Factor 8 (IRF8) is required for development, maturation and expression of anti-microbial defenses of myeloid cells. BXH2 mice harbor a severely hypomorphic allele at Irf8 (Irf8R294C) that causes susceptibility to infection with intracellular pathogens including Mycobacterium tuberculosis. We report that BXH2 are completely resistant to the development of cerebral malaria (ECM) following Plasmodium berghei ANKA infection. Comparative transcriptional profiling of brain RNA as well as chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) was used to identify IRF8-regulated genes whose expression is associated with pathological acute neuroinflammation. Genes increased by infection were strongly enriched for IRF8 binding sites, suggesting that IRF8 acts as a transcriptional activator in inflammatory programs. These lists were enriched for myeloid-specific pathways, including interferon responses, antigen presentation and Th1 polarizing cytokines. We show that inactivation of several of these downstream target genes (including the Irf8 transcription partner Irf1) confers protection against ECM. ECM-resistance in Irf8 and Irf1 mutants is associated with impaired myeloid and lymphoid cells function, including production of IL12p40 and IFNγ. We note strong overlap between genes bound and regulated by IRF8 during ECM and genes regulated in the lungs of M. tuberculosis infected mice. This IRF8-dependent network contains several genes recently identified as risk factors in acute and chronic human inflammatory conditions. We report a common core of IRF8-bound genes forming a critical inflammatory host-response network.

p53 mediates loss of hematopoietic stem cell function and lymphopenia in Mysm1-deficiency

MYSM1 is a chromatin-binding transcriptional cofactor that deubiquitinates histone H2A. Studies of Mysm1-deficient mice have shown that it is essential for hematopoietic stem cell (HSC) function and lymphopoiesis. Human carriers of a rare MYSM1-inactivating mutation display similar lymphopoietic deficiencies. However, the mechanism by which MYSM1 regulates hematopoietic homeostasis remains unclear. Here, we show that Mysm1-deficiency results in p53 protein elevation in many hematopoietic cell types. p53 is a central regulator of cellular stress responses and HSC homeostasis. We thus generated double-knockout mice to assess a potential genetic interaction between Mysm1 and p53 in hematopoiesis. Mysm1(-/-)p53(-/-) mouse characterization showed a full rescue of Mysm1(-/-) developmental and hematopoietic defects. This included restoration of lymphopoiesis, and HSC numbers and functions. These results establish p53 activation as the driving mechanism for hematopoietic abnormalities in Mysm1 deficiency. Our findings may advance the understanding of p53 regulation in hematopoiesis and implicate MYSM1 as a potential p53 cofactor.

CCDC88B is a novel regulator of maturation and effector functions of T cells during pathological inflammation

Kennedy et al. identify a mutation in coiled-coil domain containing protein 88b (Ccdc88b) that confers protection against lethal neuroinflammation during experimental cerebral malaria. CCDC88B is expressed in immune cells and regulates T cell maturation and effector functions. In humans, the CCDC88B gene maps to a locus associated with susceptibility to several inflammatory and autoimmune disorders.

Primary Immunodeficiencies and Inflammatory Disease: A Growing Genetic Intersection

Recent advances in genome analysis have provided important insights into the genetic architecture of infectious and inflammatory diseases. The combined analysis of loci detected by genome-wide association studies (GWAS) in 22 inflammatory diseases has revealed a shared genetic core and associated biochemical pathways that play a central role in pathological inflammation. Parallel whole-exome sequencing studies have identified 265 genes mutated in primary immunodeficiencies (PID). Here, we examine the overlap between these two data sets, and find that it consists of genes essential for protection against infections and in which persistent activation causes pathological inflammation. Based on this intersection, we propose that, although strong or inactivating mutations (rare variants) in these genes may cause severe disease (PIDs), their more subtle modulation potentially by common regulatory/coding variants may contribute to chronic inflammation.

USP15 regulates type I interferon response and is required for pathogenesis of neuroinflammation

Genes and pathways in which inactivation dampens tissue inflammation present new opportunities for understanding the pathogenesis of common human inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis. We identified a mutation in the gene encoding the deubiquitination enzyme USP15 (Usp15L749R) that protected mice against both experimental cerebral malaria (ECM) induced by Plasmodium berghei and experimental autoimmune encephalomyelitis (EAE). Combining immunophenotyping and RNA sequencing in brain (ECM) and spinal cord (EAE) revealed that Usp15L749R-associated resistance to neuroinflammation was linked to dampened type I interferon responses in situ. In hematopoietic cells and in resident brain cells, USP15 was coexpressed with, and functionally acted together with the E3 ubiquitin ligase TRIM25 to positively regulate type I interferon responses and to promote pathogenesis during neuroinflammation. The USP15-TRIM25 dyad might be a potential target for intervention in acute or chronic states of neuroinflammation.

Distal Limb Patterning Requires Modulation of cis-Regulatory Activities by HOX13

SUMMARY The combinatorial expression of Hox genes along the body axes is a major determinant of cell fate and plays a pivotal role in generating the animal body plan. Loss of HOXA13 and HOXD13 transcription factors (HOX13) leads to digit agenesis in mice, but how HOX13 proteins regulate transcriptional outcomes and confer identity to the distal-most limb cells has remained elusive. Here, we report on the genome-wide profiling of HOXA13 and HOXD13 in vivo binding and changes of the transcriptome and chromatin state in the transition from the early to the late-distal limb developmental program, as well as in Hoxa13−/−; Hoxd13−/−limbs. Our results show that proper termination of the early limb transcriptional program and activation of the late-distal limb program are coordinated by the dual action of HOX13 on cis-regulatory modules.

Genetics of Infectious and Inflammatory Diseases: Overlapping Discoveries from Association and Exome-Sequencing Studies

Genome technologies have defined a complex genetic architecture in major infectious, inflammatory, and autoimmune disorders. High density marker arrays and Immunochips have powered genome-wide association studies (GWAS) that have mapped nearly 450 genetic risk loci in 22 major inflammatory diseases, including a core of common genes that play a central role in pathological inflammation. Whole-exome and whole-genome sequencing have identified more than 265 genes in which mutations cause primary immunodeficiencies and rare forms of severe inflammatory bowel disease. Combined analysis of inflammatory disease GWAS and primary immunodeficiencies point to shared proteins and pathways that are required for immune cell development and protection against infections and are also associated with pathological inflammation. Finally, sequencing of chromatin immunoprecipitates containing specific transcription factors, with parallel RNA sequencing, has charted epigenetic regulation of gene expression by proinflammatory transcription factors in immune cells, providing complementary information to characterize morbid genes at infectious and inflammatory disease loci.

Specific Dysregulation of IFNγ Production by Natural Killer Cells Confers Susceptibility to Viral Infection

Natural Killer (NK) cells contribute to the control of viral infection by directly killing target cells and mediating cytokine release. In C57BL/6 mice, the Ly49H activating NK cell receptor plays a key role in early resistance to mouse cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. Here we show that transgenic expression of Ly49H failed to provide protection against MCMV infection in the naturally susceptible A/J mouse strain. Characterization of Ly49H+ NK cells from Ly49h-A transgenic animals showed that they were able to mount a robust cytotoxic response and proliferate to high numbers during the course of infection. However, compared to NK cells from C57BL/6 mice, we observed an intrinsic defect in their ability to produce IFNγ when challenged by either m157-expressing target cells, exogenous cytokines or chemical stimulants. This effect was limited to NK cells as T cells from C57BL/6 and Ly49h-A mice produced comparable cytokine levels. Using a panel of recombinant congenic strains derived from A/J and C57BL/6 progenitors, we mapped the genetic basis of defective IFNγ production to a single 6.6 Mb genetic interval overlapping the Ifng gene on chromosome 10. Inspection of the genetic interval failed to reveal molecular differences between A/J and several mouse strains showing normal IFNγ production. The chromosome 10 locus is independent of MAPK signalling or decreased mRNA stability and linked to MCMV susceptibility. This study highlights the existence of a previously uncovered NK cell-specific cis-regulatory mechanism of Ifnγ transcript expression potentially relevant to NK cell function in health and disease.

Transcriptional mechanisms that control expression of the macrophage colony-stimulating factor receptor locus

The proliferation, differentiation, and survival of cells of the macrophage lineage depends upon signals from the macrophage colony-stimulating factor (CSF) receptor (CSF1R). CSF1R is expressed by embryonic macrophages and induced early in adult hematopoiesis, upon commitment of multipotent progenitors to the myeloid lineage. Transcriptional activation of CSF1R requires interaction between members of the E26 transformation-specific family of transcription factors (Ets) (notably PU.1), C/EBP, RUNX, AP-1/ATF, interferon regulatory factor (IRF), STAT, KLF, REL, FUS/TLS (fused in sarcoma/ranslocated in liposarcoma) families, and conserved regulatory elements within the mouse and human CSF1R locus. One element, the Fms-intronic regulatory element (FIRE), within intron 2, is conserved functionally across all the amniotes. Lineage commitment in multipotent progenitors also requires down-regulation of specific transcription factors such as MYB, FLI1, basic leucine zipper transcriptional factor ATF-like (BATF3), GATA-1, and PAX5 that contribute to differentiation of alternative lineages and repress CSF1R transcription. Many of these transcription factors regulate each other, interact at the protein level, and are themselves downstream targets of CSF1R signaling. Control of CSF1R transcription involves feed-forward and feedback signaling in which CSF1R is both a target and a participant; and dysregulation of CSF1R expression and/or function is associated with numerous pathological conditions. In this review, we describe the regulatory network behind CSF1R expression during differentiation and development of cells of the mononuclear phagocyte system.